The ligase chain reaction (LCR) is a method for amplifying a specific nucleic acid sequence (target) in a sample. LCR can be used to detect single or double stranded DNA targets. Typically, two ligatable pairs of probes are employed in excess over the target, one pair of the probes are hybridizable to the other. The target DNA is first denatured (if double stranded) to allow for the hybridization of the ligatable probe pairs to their respective complementary strands. The hybridized probes are then ligated by DNA ligase. Next, the ligated probes are dissociated from the target and function as target sequences themselves. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. The process of LCR is described in the literature, including EP-A-320,308, EP-A-439,182, EP-A-336,731, WO 89/09835, WO 89/12696, and WO 90/01069 among others.
A common problem for LCR is non-specific (i.e. target independent) amplification which can lead to false positive results. This can occur, for example, when a pair of adjacent LCR probes are ligated to each other in the absence of the target. Since LCR probes are typically used in high concentration relative to the target the possibility of target independent ligation is great, and there is a comensurate need to overcome this concern.
Methods for reducing target independent ligation events have been described. For example, EP-A-439,182 describes a variation of LCR wherein one of the probes of the ligatable pair is modified so that it cannot be ligated until a correction event takes place. Correction events take place only when the probe is hybridized to target. Specifically, this application describes modifications to the 3' ends of the upstream probe, where upstream refers to the probes whose 3' ends participate in the ligation reaction. Disclosed modifications are a 3' blocking group, such as phosphate; a 3' overhang of ribonucleotides (on a deoxyribonucleotide probe); 3' overhangs including an abasic site; and 3' gaps which must be filled in to render the probes adjacent and ligatable. None of the disclosed embodiments involve modifications of the 5' end of the downstream probe.